Integrative molecular biology requires understanding interactions of large numbers of pathways. Similarly, molecular medicine increasingly relies on complex macromolecular diagnostics to guide therapeutic choices. However, genome-wide protein and mRNA copy number distributions in each cell type generally have highly skewed Pareto distributions in which the vast majority of genes have low expression levels. Critical integrative control molecules such as transcription factors and noncoding RNAs are generally expressed at low copy number. Thus contamination by more highly expressing cell phenotypes is particularly problematic in molecular analyses of pathological tissues without first isolating specific cell populations. Laser capture microdissection (LCM) of tissues provides a robust method to separate specific cell populations from complex tissues and thus allow evaluation of thousands of regulated transcription factors, cell regulators, and receptors that are expressed at low copy number. For example, in a recent collaboration with the NEI, we adapted LCM method to isolate localized (3D) cells at the site of retinal topological closure and performed microarray analysis of gene expression at 8 time points of embryonic development. This enabled us to identify low copy number transcriptions factors that when blocked lead to loss of closure in animal models. These transcription factors and approximately 200 other temporally and spatially covariant genes appear likely to play a role in coloboma, an inborn developmental defect of the retina occurring in humans. We are developing novel mathematical approaches to identify better the specific networks of genes that drive such local tissue development within such datasets. The LCM techniques that we invented sixteen years ago are now widely used in molecular analysis of genetics and gene expression changes within target cells within complex tissues. This microscope-based microdissection, which uniquely allows high-resolution imaging of the captured material before submission for downstream multiplex molecular analysis, has a proven role in research studies. However, the requirement for visual targeting has limited its applications in proteomic research where an impractical number of target decisions are required for analysis of less abundant proteins. Similarly microdissection use in clinical diagnostics has been limited by subjectivity, low through-put and cost associated with targeting of specific cells in a specialized LCM microscope. Recently, in collaboration with NCI and CIT, we invented and patented an automatic target-activated microdissection technique based on localized absorption within a uniform illuminated region by specifically stained cells. The specific staining determines the location of thermoplastic bond formation and the precision with which targets are automatically captured. This does not require user visualization or microscopic targeting. This technique combines our physical understanding of light-activated thermoplastic microbonding with standard histochemical staining techniques that provide high absorptive contrast for innumerable specific targets within tissues. For example, standard immunohistochemistry creates absorption specific to the presence of a specific antigen or protein. We irradiate the entire immunostained tissue section, but only the specifically stained targets are heated and bond to the thermoplastic polymer on its surface. The light dose needed is much less than needed in commercial laser microdissection. This reduces the thermal transients in the tissue and film and improves spatial resolution to better than 1 micron while dramatically increasing the rate at which our laser system can scan the whole section. Consequently the new method is particularly well suited to isolate highly dispersed, specific cell populations (e.g., specific neurons within a brain section) or specific organelles (e.g., nuclei of invasive cancer cells). The spatial relationships (morphology) among the specific cells in the tissue are preserved on the transfer film. Over the last two years, we (CIT, NIBIB, NICHD) have designed and built a low-cost Xenon flashlamp fTAM system that simultaneously captures all specifically stained targets within a large region of interest with one or two, 200-microsec white light pulses. The robustness and precision of this approach relies on new transfer films that we have developed with Nicole Morgan (NIBIB) which have an ultrathin (1 micron) thermoplastic polymer coating on a thermally stable, clear polymer film. The broad spectrum flashtube allows spectral filtering to be optimized for the specific stain colors. For example, we have used the standard histochemical stain, toluidine blue, with orange filtered flash to capture cancer cell nuclei from a high grade prostate cancer histology slide. In collaboration with Drs Markey, Lippincott-Schwartz, Tangrea and Morgan under an NIH Directors Challenge Award, we applied our technology to proteomics studies of subcellular organelles. We are optimizing this approach for specifically stained neuronal nuclei within formalin-fixed, paraffin-embedded brain sections. We hope to integrate our ability to microtransfer specific organelles with a variety of omic multiplex molecular analyses and thereby increase our sensitivity to molecular species associated with specific subcellular structures. For a number of years clinical molecular diagnostics have focused on discovery of concise sets of pathology specific biomakers that might be quantitatively assayed from a routinely accessible clinical sample (e.g., blood, biopsy or surgical sample). Microdissection currently has an important role in such discovery, by isolating phenotypically pure pathological samples. The utility of microdissection in clinical diagnostics will require integration with current clinical pathology methods while efficiently providing increased accuracy by analyzing many stage-specific disease markers within such purified phenotypic targets compared to the complex and variable mixtures of cell types in the original clinical specimen. For this to be practical, microdissection of clinical samples (e.g., formalin-fixed paraffin-embedded tissue sections and cytology specimens) must be reduced to a simpler, lower-cost, robust method. To meet this need, we are developing simple, robust approaches for integration of our thermoplastic microtransfer methods of microdissection with upstream pathology work-flow and downstream macromolecular analysis for clinically practical multiplex molecular diagnostics. Given the potential of such integration to more reliably measure larger numbers of interacting biomolecules with specific pathophysiological cells, we foresee an evolution of molecular diagnosis from one based on the qualitative or quantitative analysis of a few abundant, specific biomarker macromolecules to one in which special knowledge-based clustering algorithms characterize disease state with high-dimensional molecular data from microdissected samples. In summary, we are developing new technologies that integrate microdissection with macromolecular analysis of histology and cytology samples that would allow the study of many less abundantly expressed biomolecules in determining normal function and pathological changes. Our goal is to have our new technology commercialized and integrated into better molecular diagnostics to guide selection of patient appropriate clinical therapies.